Hepatitis C virus (HCV) is the predominant cause of non-A, non-B hepatitis worldwide. In this country, HCV is responsible for 100,000 to 300,000 infections annually. In half of these cases the infection becomes chronic, and 20% of patients with chronic infection will develop cirrhosis. Moreover, chronic HCV infection has been linked to the development of hepatocellular carcinoma. While initial efforts directed at the development of immunoassays have achieved success in the diagnosis of HCV infection, the variability of viral surface proteins among isolates and the absence of protective immunity in animal studies suggest that effective immunization against the virus will be difficult to accomplish. These observations reinforce the need to design antiviral therapy based on an understanding of the processes fundamental to viral replication. Of the proteins expressed by HCV and related viruses, the RNA polymerase (replicase) is likely responsible for the copying of the RNA genome. Because replicases have not been found in eukaryotic cells, HCV-infected cells could be selectively targeted for antiviral therapy directed at the protein. Early studies have demonstrated the presence of a replicase activity specific to HCV-infected crude liver extracts capable of elongating exogenously supplied HCV RNA. The aim of this proposal is to characterize the enzymatic properties of the HCV replicase. The HCV replicase will be characterized from crude liver extracts with respect to its kinetics, template specificity, cofactor requirements, and the physical features of the reaction product. Putative replicase inhibitors will be tested as a means of defining substrate specificity. Enzymatic activity will be characterized from more purified fractions using standard separation techniques as well as from recombinant sources of protein. Replicase activity will also be examined from potential extrahepatic reservoirs of HCV infection and from prospective cell culture systems. Because HCV RNA replication is likely to take place in a multiprotein replication complex, steps will be taken to isolate intact HCV replication complexes that may posses an enzymatically enriched replicase activity. Ultracentrifugation of untreated and detergent-treated crude low-salt extracts will be used to separate complexes. Active fractions will be characterized and compared to purified replicase fractions. Immunoblot techniques will be used to determine the presence of viral proteins in the replication complex. In later phases of these studies, extracts will be examined for replicase-binding proteins using a system that expresses the HCV replicase as an immobilized fusion protein. Overall, these studies aimed at characterizing the biochemical properties of HCV replicase and HCV replication complexes should yield insights into the potentially unique mechanisms underlying HCV RNA replication and facilitate the design of antiviral therapy aimed at inhibiting this virus-specific enzyme.